Dilated cardiomyopathy (DCM) is a precursor of systolic heart failure (HF), the leading cause of death and indication for cardiac transplantation in the U.S. DCM has many etiologies, including genetic predisposition. Although over 300 mutations in 60 different DCM genes have been identified, these account for a small fraction of all DCM cases. Because epidemiologic studies indicate that genetics are a major contributing factor to DCM, we suggest that new strategies are needed to define the human DCM mutations. Previously defined DCM mutations alter proteins with disparate functions, implying that many different pathways can lead to HF. To uncover the relationship between DCM mutations and pathways we will define transcriptional responses in to defined gene mutations in ventricular tissues. These studies may improve gene-based diagnosis suggest interventions that ultimately limit the progression of DCM to HF. To comprehensively define the genetic architecture of DCM we will harness new technologies and methodologies. Our approach capitalizes on a low cost method to capture and analyze large numbers of known DCM genes and harnesses next generation sequencing platforms to interrogate these. We provide preliminary data of the success of this approach. These data also indicate a distinct advantage of the proposed approach over traditional strategies, in moderate sized insertions and deletions, indels, which have previously escaped detection, were found. With continued improvement of our analysis pipeline to detect indels, we expect that resequencing of known DCM genes will reveal many more missed mutations. After this initial screen, we will harness whole exome sequencing to discovery novel DCM genes. Together these strategies will define the spectrum of DCM genes, and will the contribution of different gene mutations to the burden of DCM, information that will inform our understanding of disease mechanisms. Next generation sequencing will also be harnessed to interrogate responses activated by gene mutations. Using this platform, we devised a strategy to interrogate RNA levels and structure. We will use this approach to characterize the left ventricular transcriptome in patients with defined mutations. By incorporating pathway analyses we expect to define the signatures associated with pathways activated by different DCM genes. We have assembled a large cohort of samples with clinical data and an investigative team with extensive experience in DCM and human molecular genetics for these studies. To define the genetic architecture and pathways of DCM we propose the following aims: 1) Subgenome-capture and sequence DCM genes in 1500 DCM cases and 3000 controls; 2) Discover novel DCM genes by whole exome sequencing of mutation-negative familial DCM cases; 3) Assess the role of somatic mutation in mutation-negative sporadic DCM cases; and 4) Define RNA responses to DCM mutations in experimental models and human LV tissues. 1